A gene, LpOryzainα, that delays leaf senescence in perennial ryegrass and its application.

Abstract

The application discloses a perennial ryegrass leaf senescence gene LpOryzain alpha and application, and belongs to the technical field of plant genetic engineering.The application identifies a cysteine protease LpOryzain alpha in perennial ryegrass, and the nucleotide sequence of the CDS of the gene is shown as SEQ ID NO.9.The expression amount of LpOryzain alpha is higher with the senescence of leaves in perennial ryegrass.Further overexpression of LpOryzain alpha in Arabidopsis and perennial ryegrass shows that LpOryzain alpha can delay the senescence degree of leaves.It can be seen that the perennial ryegrass leaf senescence gene LpOryzain alpha has a function of delaying the senescence of plant leaves, and provides an important reference gene and theoretical basis for improving the quality of turf grass and pasture.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a gene LpOryzainα that delays leaf senescence in perennial ryegrass and its application. Background Technology

[0002] Leaves play a crucial role in photosynthesis and respiration in plants, and are essential for substrate conversion and energy metabolism. Senescence is a degenerative process at the cellular, tissue, organ, or organismal level, ultimately leading to death and the end of the plant's life cycle. Leaf senescence falls under the category of organ-level senescence in plants and is often closely accompanied by cell or organismal death. When plants enter the reproductive growth stage, such as the grain-filling and ripening stages, annual crops will enter leaf senescence, eventually leading to the death of the entire plant, thus completing their life cycle. For perennial plants, leaf senescence often stems from seasonal changes and damage caused by biotic and abiotic stresses. Premature leaf senescence shortens the vegetative growth period, accelerates the transition from the vegetative to the reproductive stage, reduces the plant's nutritional capacity, and negatively impacts plant productivity.

[0003] Cystine proteinases (CysPs), also known as thiol proteases, are important proteases widely found in animals, microorganisms, and plants. Their active sites contain Cys residues, and the active sites of these enzymes are generally identified through inhibitors. Their catalytic mechanisms typically involve a catalytic triad (Cys, His, Asn) or a similar catalytic pair. CysPs were first isolated and characterized from papaya (Carica papaya) by Gopal Chunder Roy in 1873. A representative member is the C1 subfamily papain, which is the most extensively studied cysteine ​​protease in plants. In addition, CysP has 5 subfamilies: (1) The C2 subfamily is calcium-dependent cysteine ​​proteases (calpains), which are a class of polypeptides that do not contain signal peptides and are mainly cytoplasmic proteins; (2) The C12 subfamily is ubiquitin C-terminal hydrolases, all of which are synthesized in the absence of propeptides and remain in the cell, and are highly selective for hydrolyzing the bonds formed by ubiquitin C-terminal Gly; (3) The C13 subfamily is vacuole processing enzymes (VPEs), which belong to the legume aspartic protease family and are mainly used to process precursor proteins and degrade proteins in vacuoles. In addition, some VPEs also regulate programmed cell death in plant development and defense responses through their aspartic protease activity; (4) The C14 subfamily belongs to aspartic acid-specific cysteine ​​proteases, which are widely present in prokaryotic and eukaryotic cells and contain catalytic Cys and His residues. It is known that the apoptosis cascade is mainly controlled by caspases of the C14 subfamily; (5) The C15 subfamily is pyroglutamyl peptidase I (PGP-1). CysPs proteases have been reported to participate in a variety of protein hydrolysis and physiological processes in plants, such as senescence, tissue abscission, programmed cell death, fruit ripening, pollen development, and mobilization of stored proteins in seeds and tubers. Currently, several genes encoding cysteine ​​proteases have been cloned in various senescent plant tissues. For example, the Arabidopsis thaliana SAG12 gene encodes a cysteine ​​protease that is specifically expressed during leaf senescence and is considered a marker gene for leaf senescence; in Populus tomentosa, the cysteine ​​protease gene PtoCP1 participates in leaf senescence regulated by plant hormones. Oryzainα was first isolated from rice as a cysteine ​​protease. It possesses the highly conserved CysP functional domain Pept_C1 and belongs to the papain-like cysteine ​​protease class. Existing studies have reported that Oryzainα is associated with rice blast disease, but whether Oryzainα participates in the regulation of leaf senescence remains unknown.

[0004] Perennial ryegrass (Lolium perenne L.) belongs to the genus Lolium in the family Poaceae. It is characterized by its well-developed fibrous root system, abundant tillering, rapid turf establishment, and strong resistance to diseases and pests, making it an important pioneer grass species for lawn establishment. Furthermore, perennial ryegrass exhibits strong regeneration ability and high yield after mowing, making it an important cultivated forage and green manure crop. However, under normal conditions, perennial ryegrass leaves age rapidly, with each tiller having only three green leaves. It is also highly susceptible to abiotic stresses such as high temperature, drought, shade, and rust, leading to accelerated leaf senescence. This severely impacts the green period of lawns and the yield and quality of forage, becoming a significant factor restricting the development of my country's ryegrass industry. Therefore, researching key genes regulating leaf senescence in ryegrass is of great significance for improving the quality of turfgrass and the production efficiency of forage, as well as ensuring the security of the national turf and forage industries. Summary of the Invention

[0005] The purpose of this invention is to provide a gene LpOryzainα that delays leaf senescence in perennial ryegrass and its application, in order to solve the problems existing in the prior art. By overexpressing LpOryzainα in Arabidopsis thaliana or perennial ryegrass, leaf senescence in Arabidopsis thaliana or perennial ryegrass can be significantly delayed.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a perennial ryegrass gene LpOryzainα that delays leaf senescence, the CDS sequence of which is shown in SEQ ID NO.9.

[0008] The present invention also provides a protein encoded by the LpOryzainα, the amino acid sequence of which is shown in SEQ ID NO. 10.

[0009] The present invention also provides a recombinant vector comprising the LpOryzainα described above.

[0010] The present invention also provides recombinant bacteria comprising the recombinant vector described above.

[0011] This invention also provides the use of the LpOryzainα, the protein, the recombinant vector, or the recombinant bacteria in any of the following:

[0012] (1) Application in delaying plant leaf senescence;

[0013] (2) Application in the cultivation of transgenic plants that delay leaf senescence;

[0014] (3) Application in increasing the chlorophyll content of plants.

[0015] Preferably, overexpression of the LpOryzainα gene increases chlorophyll content in plants and / or delays leaf senescence.

[0016] Preferably, the plant includes Arabidopsis thaliana or perennial ryegrass.

[0017] The present invention also provides a method for delaying plant leaf senescence, comprising the step of overexpressing the LpOryzainα in the plant.

[0018] The present invention also provides a method for cultivating transgenic plants that delay leaf senescence, comprising the step of overexpressing the LpOryzainα in plants to construct transgenic plants that delay leaf senescence.

[0019] Preferably, the plant includes Arabidopsis thaliana or perennial ryegrass.

[0020] The present invention discloses the following technical effects:

[0021] This invention identified a cysteine ​​protease, LpOryzainα, in perennial ryegrass, with a gene length of 1392 bp. qRT-PCR analysis revealed that LpOryzainα is induced by the plant hormone ABA. Furthermore, the expression level of LpOryzainα in perennial ryegrass increases with leaf senescence. Further overexpression of LpOryzainα in Arabidopsis thaliana and perennial ryegrass showed that LpOryzainα can delay leaf senescence.

[0022] The LpOryzainα gene, a perennial ryegrass leaf senescence gene, has the function of delaying leaf senescence in plants, providing an important reference gene and theoretical basis for improving the quality of turfgrass and forage grasses. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 For LpOryzainα expression profile analysis; A: LpOryzainα response to different plant hormones (H2O is the control); B: LpOryzainα expression in different tissues; C: LpOryzainα expression in transcriptome data at different leaf development stages;

[0025] Figure 2Phylogenetic analysis of the LpOryzainα gene in perennial ryegrass with other species; (delayed senescence: delaying leaf senescence).

[0026] Figure 3 Amino acid sequence comparison of the perennial ryegrass aging gene LpOryzainα with other species;

[0027] Figure 4 Predicted protein domain structure for the LpOryzainα gene, a gene for delaying the aging of perennial ryegrass.

[0028] Figure 5 A diagram illustrating the construction process of the recombinant vector pEarlyGate103-LpOryzainα;

[0029] Figure 6 Phenotypic analysis of leaf senescence induced by dark treatment in Arabidopsis LpOryzainα overexpression lines; A: Phenotypic of LpOryzainα transgenic Arabidopsis after dark treatment; B: Detection of LpOryzainα transcription level in different transgenic Arabidopsis lines; CD: Changes in chlorophyll fluorescence and chlorophyll content before and after dark treatment, respectively; WT represents wild-type lines; L1, L2, L3, L4, and L5 represent overexpression lines; DAD represents the number of days of dark treatment.

[0030] Figure 7 Identification of senescence phenotype in LpOryzainα overexpressing lines of perennial ryegrass; A: Phenotype of LpOryzainα detached leaves after dark treatment; B: Transcriptional level detection of LpOryzainα in the same transgenic line; C: GUS tissue staining; D: DNA level detection in transgenic ryegrass, with markers representing standard DNA molecules; EF: Changes in chlorophyll fluorescence and chlorophyll content before and after dark treatment, respectively; In the figure, WT represents wild-type lines, L2 and L5 represent overexpressing lines, and DAD represents the number of days of dark treatment. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Example 1: LpOryzainα gene expression pattern

[0037] 1. Material processing of perennial ryegrass

[0038] The inventors selected perennial ryegrass (BV) as the test material (from the Chengdu campus of Sichuan Agricultural University) and performed the following treatment:

[0039] a) First, germinate the seeds in a petri dish. Once seedlings emerge (8-10 days), transfer them to a hydroponic system. After transferring to a hydroponic box, cultivate them using Hoagland nutrient solution (Hoagland Biotechnology Co., Ltd.), changing the nutrient solution every three days. When two-thirds of the fifth leaf (counting from top to bottom) turns yellow, take samples from the first to fifth leaves, as well as nine parts: root, stem, crown, and leaf sheath.

[0040] b) After hydroponically cultivating perennial ryegrass using the above method for 3 weeks, treat it using a spray method. Specifically, treat it with abscisic acid (ABA), gibberellin (GA), jasmonic acid (JA), salicylic acid (SA), auxin (IAA), ethylene (ETH), 6-benzylaminopurine (6-BA), water (H2O), brassinolide (BR), and strigolactone (SL). Then take materials at 0h and 1h, 2h, 4h, 8h, 12h, and 24h after treatment. Take 5 samples for each treatment at each time point and store them at -80℃.

[0041] c) Transplant the seedlings that germinated in the petri dishes into nutrient soil. After about 2 weeks of growth, mark the newly grown branches and shoots as day 0. Then, take leaves at days 4, 12, 20, 28, and 36 for transcriptome sequencing. A large number of samples are required.

[0042] 2. Relative expression level analysis

[0043] RNA extraction was performed using the plant total RNA extraction kit from Magen Biotech Ltd., following the kit's instructions. After RNA extraction, cDNA was synthesized via reverse transcription. The relative expression levels of LpOryzainα in different tissues and under different treatments were determined using quantitative real-time polymerase chain reaction (qPCR). eIF4A was used as an internal control gene. The primers used in the qPCR were as follows:

[0044] qLpOryzainα-F: 5'-GGGAGTTGCTGGCTTTCT-3' (SEQ ID NO.1)

[0045] qLpOryzainα-R: 5'-CGGAGTCGATTCCACCATTGT-3' (SEQ ID NO.2)

[0046] LpeIF4A-F: 5'-AACTCAACTTGAAGTGTTGGAGTG-3' (SEQ ID NO.3)

[0047] LpeIF4A-R: 5'-AGATCTGGTCCTGGAAAGAATATG-3' (SEQ ID NO.4)

[0048] The qRT-PCR reaction system was 20 μL, containing 5 μL cDNA, 0.4 μL qLpOryzainα-F, 0.4 μL qLpOryzainα-R, 10 μL SYBR enzyme, and the remainder was added to 20 μL with RNase-free ddH2O.

[0049] The qRT-PCR reaction program was: 95℃ for 30 s; 95℃ for 5 s, 58℃ for 30 s, 40 cycles; the melting curve was set to the machine's default program. 2 -ΔΔCt The method calculates the relative expression level of genes.

[0050] Test results as follows Figure 1 As shown, LpOryzainα is highly expressed in senescent leaves. Figure 1 (B), and its expression level increased with leaf senescence in the transcriptome data, reaching a peak on day 28. Figure 1 (C); and different treatment results showed that it was ABA-induced ( Figure 1 (A). The above results indicate that LpOryzainα may play an important role in the regulation of leaf senescence.

[0051] 3. Verification of the LpOryzainα gene

[0052] 3.1 Cloning of LpOryzainα

[0053] Using perennial ryegrass cDNA as a template, PCR amplification was performed using the 5'UTR upstream primer F1 (LpOryzainα-F) and the 3'UTR downstream primer R1 (LpOryzainα-R). Amplification was performed using the Q5 High-Fidelity DNA Polymerase kit. CDS region-specific primers (LpOryzainα-CDS-F and LpOryzainα-CDS-R) were redesigned, and the recovered PCR product from the first PCR was used as a template for a second round of amplification.

[0054] The primers required for PCR amplification are as follows:

[0055] LpOryzainα-F: 5'-CCCATCTACCACTCTCCCCT-3' (SEQ ID NO.5);

[0056] LpOryzainα-R: 5'-AGTCTTTACGCGCTGCTCTT-3' (SEQ ID NO. 6);

[0057] LpOryzainα-CDS-F: 5'-ctagacccggggaattcATGAGGACCTCCACGGCT-3' (SEQ IDNO.7);

[0058] LpOryzainα-CDS-R: 5'-tcgacactagtaagcttCGCGCTGCTCTTCATGCCGT-3' (SEQ ID NO. 8).

[0059] In the sequences shown in SEQ ID NO.7 and SEQ ID NO.8, the lowercase letters represent the amplified sequences of the restriction enzyme sites.

[0060] The PCR reaction system consisted of: 4 μL 5×Q5 Reaction Buffer, 0.4 μL 10 mM dNTPs, 1 μL cDNA, 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 0.2 μL Q5 High-Fidelity DNA Polymerase, 4 μL 5×Q5 High GC Enhancer (optional), and ddH2O to a final volume of 20 μL.

[0061] The PCR reaction conditions were: 98℃ for 30s; 98℃ for 10s, 68℃ for 30s, 72℃ for 1min, 30 cycles; 72℃ for 2min, and stored at 10℃.

[0062] The amplified CDS sequence fragment was digested with enzymes and then ligated into the pEntry vector using T4 ligase to obtain pEntry-LpOryzainα. This was then sent to a sequencing company for sequencing, and the results were compared with the reference genome sequence of perennial ryegrass. Based on the obtained amino acid sequence, homologs of LpOryzainα in other species were searched using NCBI, and a phylogenetic tree was constructed (see [link to citation]). Figure 2 The obtained homologous gene amino acid sequences were compared with LpOryzainα multiple sequence sequences, and it was found that both contained the papain-like cysteine ​​protease domain Pept_C1 region (see...). Figure 3 The predicted domain diagram of the protein encoded by LpOryzainα is shown below. Figure 4 .

[0063] LpOryzainα is a cysteine ​​protease. The gene is 1392 bp long and encodes 463 amino acids.

[0064] The CDS sequence (SEQ ID NO.9) of the amplified LpOryzainα gene is as follows:

[0065]

[0066] The amino acid sequence of the protein encoded by the gene (SEQ ID NO.10):

[0067] MRTSTALLAAAAALLLSLAAAADMSIVSYGERSEEEARRMYAEWKAEHGRNSNAIGGEDELRFAVFRDNLRYVDEHNAAADAGVHSFRLGLNRFADLTNEEYRSTYLGVRTKPERK QKLSSRYQAAVNEDLPESVDWRTKGAVPAVKDQGGCGSCWAFSAIAAVEGINAIATGDLIALSEQELVDCDTSYNEGCNGGLMDYAFEFIINNGGIDSEEDYPYTEKDGRCDANKK NAKVVTIDGYEDVPVNSEKSLQKAVANQPISVAIEAGGRAFQLYSSGIFTGTCGTALDHGVAAVGYGTEDGKDYWIVRNSWGSSWGEAGYVRMERNIKSTDGKCGIAIEPSYPLKT GANPPNPGPTPPAPVPPTPPSSVCDSYYTCPASTTCCCIFEYGKECFAWGCCPLEGASCCDDHYSCCPHDYPVCNTRRGTCSATKDSPLSVKALKRIMATRTGARRAEDGMKSSA.

[0068] 3.2 Construction of recombinant expression vector and recombinant bacteria

[0069] a) First, the above pEntry-LpOryzainα plasmid was digested with PvuI restriction endonuclease.

[0070] The enzyme digestion PCR reaction system was as follows: 4 μL Cut Buffer, 1 μL PvuI restriction enzyme, 2 μL pEntry-LpOryzainα, and ddH2O to a final volume of 40 μL.

[0071] The enzyme digestion PCR reaction program was: 37℃, 70 min.

[0072] b) The above enzyme digestion products were then recovered as plasmids, and LR recombinase was used to perform LR recombination with the pEearlyGate103 expression vector to form a new recombinant expression vector pEarlyGate103-LpOryzainα (see [link to original text]). Figure 5 ).

[0073] The recombinant PCR reaction system consisted of: 0.5 μL LR mix, 3 μL pEarlyGate103, 0.5 μL enzyme digestion product, and ddH2O to a final volume of 5 μL.

[0074] The recombinant PCR reaction procedure was as follows: after reacting at 25℃ for 1 h, add 0.5 μL proteinase K and incubate at 37℃ for 10 min.

[0075] c) The plasmid was recovered using the enzyme digestion product described in a), and then LR recombinase was used to perform LR recombination with the Pcambial1305.2 expression vector to form a new recombinant expression vector pCambial1305.2-LpOryzainα.

[0076] The specific method is the same as that used for constructing the pEarlyGate103-LpOryzainα vector.

[0077] d) Transform the above pEarlyGate103-LpOryzainα expression vector into GV3101 Chemically Competent Cell (Shanghai Weidi Biotechnology Co., Ltd.) according to the instructions.

[0078] The specific operating method is as follows:

[0079] ① Allow Agrobacterium, which is stored at -80℃, to partially melt at room temperature for a short while, and then insert it into ice while it is in a state of ice-water mixture.

[0080] ② Take 20 μL of Agrobacterium and add 2 μL of pEarlyGate103-LpOryzainα plasmid to it. Mix well by tapping the bottom of the tube with your hand. Incubate sequentially on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0081] ③ Add 700 μL of antibiotic-free LB and incubate at 28°C with shaking for 2–3 hours.

[0082] ④ Centrifuge at 6000 rpm for one minute to collect the bacteria. Take about 100 μL of supernatant, gently pipette and resuspend the bacterial block, and spread it on LB solid medium containing Kan (50 mg / L) and rifampicin (50 mg / L). Invert the medium and incubate at 28℃ for 2-3 days.

[0083] Finally, single colonies were selected and cultured in liquid LB medium containing Kans and rifampin at 28°C and 200 rpm for 24 hours. Afterward, bacterial PCR was performed, and positive single colonies were identified as host cells. The host cells were then stored at -80°C with glycerol.

[0084] 4. Obtaining and identifying overexpression lines

[0085] 4.1 Acquisition and Identification of Arabidopsis Strains

[0086] Arabidopsis thaliana cultivation: Wild-type seeds were sown on 1 / 2 MS medium. After vernalization for two days, they were placed in a plant growth chamber with 14 hours of light per day and 40-60% humidity for about 10 days. Then, they were transplanted into flowerpots (9cm×9cm×11cm), with 4 seedlings per pot. They were cultured in a plant growth chamber; after transplanting, they were cultured using MS nutrient solution.

[0087] The *Agrobacterium* strain carrying the pEarlyGate103-LpOryzainα vector was cultured to an OD value of 0.8. Before inoculation, the surfactant silwet-L77 was added, mixed, and incubated in the dark at room temperature for 2 hours. After removing the pods and flowers from the *Arabidopsis* plants, the aboveground parts were immersed in the host cell suspension for 40-50 seconds. Once the *Arabidopsis* seeds matured, they were harvested, and positive *Arabidopsis* were selected on 1 / 2 MS + Basta medium.

[0088] The received T0 generation Arabidopsis seeds were spread evenly on 1 / 2 MS solid medium containing 20 mg / L glufosinate. After about two weeks of growth, the healthy green seedlings were transferred to nutrient soil for further cultivation. When the Arabidopsis leaves grew to a slightly larger size, genomic DNA was extracted from the transgenic Arabidopsis for PCR identification (see...). Figure 6 (B). Simultaneously, the obtained transgenic positive lines (leaf phenotype see...) Figure 6 A) Continue to cultivate, collect its T1 generation seeds, screen for lines with a segregation ratio close to 3:1 and collect the seeds.

[0089] 4.2 Obtaining and Identifying Transgenic Lines of Perennial Ryegrass

[0090] First, the Agrobacterium carrying the pCambial1305.2-LpOryzainα vector was cultured to an OD value of 0.8. Then, the embryogenic callus of perennial ryegrass (BV) was transformed by Agrobacterium-mediated transformation to integrate the target gene into the ryegrass genomic DNA. Finally, the seedlings differentiated and grown from the callus (when the roots have grown to about 2-3 cm) were transplanted into the soil to grow.

[0091] The mature plants were identified using GUS staining to detect positive plants. Figure 7 (C)

[0092] Next, gDNA was extracted from transgenic lines and wild-type ryegrass for PCR identification. Figure 7 D),

[0093] The primers required for PCR amplification are as follows:

[0094] HIPII F: 5'-CAAACTGTGATGGAGGACACCG-3'; (SEQ ID NO. 11);

[0095] HIPII R: 5'-TATATGCTCAACACATGAGCG-3'; (SEQ ID NO. 12).

[0096] The PCR reaction system was as follows: 10 μL 2×Taq PCR MasterMix, 1 μL HIPII F, 1 μL HIPII R, 1 μL gDNA, and ddH2O added to a final volume of 20 μL.

[0097] The PCR reaction program was as follows: 94℃ for 3 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 5 min, and stored at 10℃.

[0098] like Figure 7 As shown in Figure BC, GUS staining and PCR identification confirmed that lines L2 and L5 were positive plants (see leaf phenotype). Figure 7 (A)

[0099] 4.3 Functional validation of LpOryzainα overexpression lines

[0100] a) Experimental methods

[0101] Arabidopsis thaliana: T2 generation transgenic Arabidopsis thaliana seeds and wild-type seeds were spread evenly on 1 / 2 MS solid medium containing 20 mg / L glufosinate. When the Arabidopsis thaliana had 2-3 cotyledons, they were transplanted into flowerpots for soil cultivation. Approximately 7 pots were transplanted for each line, with 4 plants per pot. After 2-3 weeks of cultivation, the experiment was conducted. First, leaves of similar growth, size, and location were cut, and a large number of replicates (approximately 20 leaves) were taken. The leaves were placed in square petri dishes with filter paper, and photographs were taken for recording. Then, the leaves were treated in the dark at room temperature, while maintaining a humid environment (to prevent the leaves from drying out or becoming waterlogged). Chlorophyll fluorescence and chlorophyll content were measured on day 0. The leaf phenotype was observed and compared with the wild type every day during the experiment. When there was a significant difference in phenotype between the two, photographs were taken, and the indicators were measured.

[0102] Perennial ryegrass: Leaves of similar growth, size, and location from the same part of the plant are selected, generally the 2nd-3rd leaves (counting from top to bottom), approximately 10 leaves in total. The leaves are placed in a humid environment and photographed for record-keeping. Then, they are treated at room temperature in the dark. Chlorophyll fluorescence and chlorophyll content are measured on day 0. Leaf phenotype is observed daily and compared with the wild type. When a significant difference in phenotype is observed, photographs are taken and the relevant indicators are measured.

[0103] The determination of physiological indicators includes the following procedures.

[0104] Chlorophyll fluorescence measurement: After the leaves were placed in the dark for 20-30 minutes, the maximum photochemical efficiency (Fv / Fm) was measured using a Pocket PEA plant chlorophyll fluorescence efficiency meter. Repeat the measurement as many times as possible.

[0105] Chlorophyll determination: Weigh 0.1g of leaf material, place it in a 2mL centrifuge tube, and add 2mL of dimethyl sulfoxide to completely immerse the leaf in the liquid. Place in the dark at room temperature. When the leaf material turns completely white, transfer 200μL of the extract to an ELISA plate and measure the absorbance of the chlorophyll extract at wavelengths of 663nm and 645nm using an ELISA reader. Then, use tweezers to place the soaked leaf material into a small envelope and dry it in an oven (65℃) until constant weight. Measure its dry weight (W).

[0106] Total chlorophyll content (mg / g) = (20.2OD) 645 +8.02OD 663 )*200 / (W*1000).

[0107] b) Experimental Results

[0108] like Figure 6 Chinese CD and Figure 7 As shown in Figure EF, under dark treatment conditions, both Arabidopsis thaliana and perennial ryegrass overexpression lines exhibited a significantly delayed chlorosis phenotype compared to the wild type. Measurements of chlorophyll fluorescence and chlorophyll content revealed that the overexpression lines had higher chlorophyll fluorescence and chlorophyll content. This indicates that overexpression of LpOryzainα significantly delayed leaf senescence.

[0109] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Perennial ryegrass genes LpOryzain α Application in any of the following: (1) Application in delaying the senescence of plant leaves; (2) Application in the cultivation of transgenic plants that delay leaf senescence; (3) Application in increasing the chlorophyll content of plants; Through overexpression LpOryzain α Genes that increase chlorophyll content in plants and / or delay leaf senescence; The LpOryzain α The CDS sequence of the gene is shown in SEQ ID NO.9; The plant in question is Arabidopsis thaliana or perennial ryegrass.

2. Perennial ryegrass genes LpOryzain α The encoded protein is used in any of the following: (1) Application in delaying the senescence of plant leaves; (2) Application in the cultivation of transgenic plants that delay leaf senescence; (3) Application in increasing the chlorophyll content of plants; Through overexpression LpOryzain α Genes that increase chlorophyll content in plants and / or delay leaf senescence; The LpOryzain α The CDS sequence of the gene is shown in SEQ ID NO.9; The plant in question is Arabidopsis thaliana or perennial ryegrass.

3. Contains genes from perennial ryegrass. LpOryzain α The recombinant vector may be used in any of the following applications: (1) Application in delaying the senescence of plant leaves; (2) Application in the cultivation of transgenic plants that delay leaf senescence; (3) Application in increasing the chlorophyll content of plants; Through overexpression LpOryzain α Genes that increase chlorophyll content in plants and / or delay leaf senescence; The LpOryzain α The CDS sequence of the gene is shown in SEQ ID NO.9; The plant in question is Arabidopsis thaliana or perennial ryegrass.

4. Contains genes from perennial ryegrass. LpOryzain α The application of recombinant bacteria in any of the following: (1) Application in delaying the senescence of plant leaves; (2) Application in the cultivation of transgenic plants that delay leaf senescence; (3) Application in increasing the chlorophyll content of plants; Through overexpression LpOryzain α Genes that increase chlorophyll content in plants and / or delay leaf senescence; The LpOryzain α The CDS sequence of the gene is shown in SEQ ID NO.9; The plant in question is Arabidopsis thaliana or perennial ryegrass.

5. A method for delaying the senescence of plant leaves, characterized in that, Including overexpression in plants LpOryzain α The steps of gene generation; The LpOryzain α The CDS sequence of the gene is shown in SEQ ID NO.9, and the plant is Arabidopsis thaliana or perennial ryegrass.

6. A method for cultivating transgenic plants that delay leaf senescence, characterized in that, Including overexpression in plants LpOryzain α The steps involved in constructing transgenic plants that delay leaf senescence, including gene editing. LpOryzain α The CDS sequence of the gene is shown in SEQ ID NO.9, and the plant is Arabidopsis thaliana or perennial ryegrass.

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